1. Field of the Invention
The present invention broadly relates to the field of medicine. More specifically, the present invention relates to the fields of medical imaging, diagnostics, and pharmaceutical therapy. The present invention provides methods and compositions for medical imaging, evaluating intracellular processes, radiotherapy of intracellular targets, and drug delivery by the use of novel cell membrane-permeant peptide conjugate coordination and covalent complexes having target cell specificity. The present invention also provides kits for conjugating radionuclides and other metals to the peptide coordination complexes.
2. DESCRIPTION OF RELATED ART
Radiopharmaceuticals in Diagnosis and Therapy
Radiopharmaceuticals provide vital information that aids in the diagnosis and therapy of a variety of medical diseases (Hom and Katzenellenbogen, Nucl Med. Biol. 24:485-498, 1997). Data on tissue shape, function, and localization within the body are relayed by use of one of the various radionuclides, which can be either free chemical species, such as the gas 133Xe or the ions 123Hxe2x88x92 and 20T1xe2x88x92, covalently or coordinately bound as part of a larger organic or inorganic moiety, the images being generated by the distribution of radioactive decay of the nuclide. Radionuclides that are most useful for medical imaging include 11C (txc2xd 20.3 min), 13N (txc2xd9.97 min), 15 (txc2xd 2.03 min), 18F (txc2xd 109.7 min), 64Cu (txc2xd 12 h), 68Ga (txc2xd 68 min) for positron emission tomography (PET) and 67Ga (txc2xd 68 min), 99mTc (txc2xd 6 h), 123I (txc2xd 13 h) and 201T1 (txc2xd 73.5 h) for single photon emission computed tomography (SPECT) (Hom and Katzenellenbogen, Nucl. Med. Biol. 24:485-498, 1997).
SPECT and PET imaging provide accurate data on radionuclide distribution at the desired target tissue by detection of the gamma photons that result from radionuclide decay. The high degree of spatial resolution of modem commercial SPECT and PET scanners enables images to be generated that map the radionuclide decay events into an image that reflects the distribution of the agent in the body. These images thus contain anatomic and functional information useful in medical diagnosis. Similarly, if the radionuclides decay in such a manner as to deposit radiation energy in or near the target cells or tissues, the same approach would enable therapeutically relevant doses of radioactivity to be deposited within the tissues.
Many radiopharmaceuticals have been prepared whose tissue localizing characteristics depend on their overall size, charge, or physical state (Hom and Katzenellenbogen, Nucl. Med. Biol. 24:485-498, 1997). Other radiopharmaceuticals are synthesized with the intention to be ligands for specific hormone, neurotransmitter, cell surface or drug receptors, as well as specific high affinity transport systems or enzymes. As these receptors and enzymes are known to be involved in the regulation of a wide variety of vital bodily functions, effective imaging agents can be used in the diagnosis or staging of a variety of disease states, in which such receptors are functioning abnormally or are distributed in an abnormal fashion, or in the monitoring of therapy (Hom and Katzenellenbogen, Nucl. Med. Biol. 24:485-498, 1997). Effective therapeutic agents can also be used to deliver pharmacologically active doses of compounds to the same receptors and enzymes.
Recent advances in molecular, structural and computational biology have begun to provide insights in the structure of receptors and enzymes that should be considered in the design of various ligands. Two key issues derived from the structure and distribution of these receptors have a direct impact on the development of new radiopharmaceuticals: 1) the location of a receptor or enzyme activity in the body (i.e., peripheral sites versus brain sites), and 2) its subcellular location (i.e., on the cell surface versus intracellular) will determine whether a radiopharmaceutical injected intravenously will need to traverse zero, one , two or more membrane barriers to reach the target. The structure of the receptor and the nature of its interaction with the ligand will determine the degree to which large ligands or ligands with large substituents may be tolerated (Hom and Katzenellenbogen, Nucl. Med. Biol. 24:485-498, 1997). For example, radiopharmaceuticals which target cell surface receptors will encounter no membrane barriers to reach their target. Natural ligands for these receptors can be large, and often are charged and, consequently, large radiopharmaceuticals are tolerated. Conversely, for a radiopharmaceutical to reach intracellular receptors or enzymes, at least one membrane barrier, the cell plasma membrane, must be traversed, and if the target site is within the central nervous system, the radiopharmaceutical must also traverse the plasma membranes of endothelial cells of the brain which constitute the blood-brain barrier. Such a situation usually favors radiopharmaceutical designs that strongly minimize ligand size and molecular weight (Hom and Katzenellenbogen, Nucl. Med. Biol. 24:485-498, 1997). Thus, as the number of membrane barriers increases, a premium is placed on keeping the size of a conventional radiopharmaceutical small ( less than 600 Da) and the lipophilicity intermediate (characterized by an octanol-water partition coefficient, log P xcx9c2) to enable the agent to traverse membranes (Dishino, et al., J Nucl Med 24:1030-1038, 1983; Papadopoulos, et al., Nucl Med Biol 20:101-104, 1993; Eckelman, Eur J Nucl Med 22:249-263, 1995). This has conventionally precluded the use of peptide radiopharmaceuticals for intracellular targets.
There has been a great deal of research into the development of radiopharmaceuticals directed toward cell surface receptors whose natural ligands are peptides. Tc-labeled peptides can span the spectrum of size. The derivatizing group or chelation core of smaller peptides has been reported to impact the in vitro binding and in vivo distribution properties of these compounds (Babich and Fischman, Nucl Med Biol 22:25-30, 1995; Liu, et al., Bioconj Chem 7:196-202, 1996). For larger peptides or proteins, the labeling process can usually occur at one or more of several reactive sites, and thus, the final mixture of compounds is less chemically defined. Thus, for larger proteins, it is usually much less clear which of these sites, if any, might be more favorable for receptor interaction and whether or not specific labeling would increase biological activity of the agent (Hom and Katzenellenbogen, Nucl. Med. Biol. 24:485-498, 1997).
It is known that low molecular weight peptides and antibody fragments provide rapid tumor targeting and uniform distribution in tumor tissues (Yokota et al., Cancer Res 53:3776-3783, 1993). While such characteristics render low molecular weight peptides attractive vehicles for the delivery of radioactivity to tumor tissues and organs for both targeted imaging and radiotherapy, nonetheless problems have been encountered. High and persistent localization of the radioactivity is observed in the kidneys, which compromises tumor visualization in the kidney region and limits therapeutic potential (Buijs, et al., J Nucl Med 33:1113-1120, 1992; Baum, et al., Cancer (Phila) 73:896-899, 1994; Choi, et al., Cancer Res 55:5323-5329, 1995; Behr, et al., J Nucl Med 36:430-441, 1995). As discussed by Arano, et al. (Cancer Res 59:128-143, 1999), radiolabeled low molecular weight peptides and antibody fragments would become much more useful for targeted imaging and therapy if the renal radioactivity levels could be reduced without impairing those in the target tissue. Previous studies have indicated that radiolabeled low molecular weight peptides and antibody fragments are likely resorbed by proximal tubules via luminal endocytosis after glomerular filtration (Silberbagl, S. Physiol Rev 68:811-1007, 1988). The long residence times of the radiometabolites generated after lysosomal proteolysis of the radiolabeled fragments in renal cells were also reported to be responsible for the persistent renal radioactivity levels (Choi, et al., Cancer Res 55:5323-5329; Rogers, et al., Bioconjugate Chem 7:511-522, 1996).
There exists a continued need for peptide-based radiopharmaceuticals that are rapidly cleared and target intracellular receptors or enzyme activities.
Peptide-Based Metal Coordination Complexes
Small peptides can be readily prepared by automated solid phase peptide synthesis (Merifield et al., Biochemistry 21:5020-5031, 1982; Houghten, Proc Natl Acad Sci USA 82:5131-5135, 1985; Lin, et al., Biochemistry 27:5640-5645, 1988) using any one of a number of well known, commercially available automated synthesizers, such as Applied Biosystems ABI 433A peptide synthesizer. Many combinations of natural and non-natural amino acids and peptide sequence mimetics (peptidomimetics) are possible, and selective engineering of favorable target-binding and pharmacokinetic properties can be accomplished with natural and unnatural peptides (Lister-James et al., Q. J Nucl. Med., 41:111-118, 1997). Peptidomimetics are unnatural biopolymers that do not contain xcex1-amino acids, but rather incorporate backbone structures with hydrogen-bonding groups (such as urea), chiral centers, side chain functionalities, and a sufficient degree of conformational restriction to behave similar to, or mimic the bioactivities of, a natural polypeptide. Peptide-based imaging agents are also well known (Lister-James et al., Q. J Nucl. Med., 41:111-118, 1997; Lister-James et al., J Nucl. Med., 38:105-111, 1997), especially those that incorporate Tc-99m as the radionuclide, the most commonly used isotope in medical imaging.
The metallic character of Tc-99m requires that it be stabilized by a chelation system to be coupled to an imaging agent. This chelator may typically involve a multiple heteroatom coordination system, or the formation of a non-labile organometallic species. There are two broad strategies for binding metals for biological applications. These are xe2x80x9cthe pendant approachxe2x80x9d and xe2x80x9cthe integrated approach,xe2x80x9d which have been recently reviewed by Katzenellenbogen and colleagues (Hom and Katzenellenbogen, Nucl. Med. Biol., 24:485-498, 1997). The pendant (or conjugate) approach involves the strategic placement of a Tc-99m-chelator-tether moiety at a site on the ligand that will not hinder binding of the ligand to its high affinity receptor. The integrated approach replaces a component of a known high-affinity receptor ligand with the requisite Tc-99m chelator such that there is a minimal change in the size, shape, structure, and binding affinity of the resultant molecule. Applications involving peptide-based imaging agents typically use the conjugate design, whereby an appropriate metal chelating moiety is affixed to the amino or carboxy terminus of the targeting peptide.
A variety of metal chelation systems have been developed for synthesis of radioisotopic and magnetic resonance peptide-based imaging agents. Peptide-based agents target extracellular or externally oriented membrane bound receptors (Hom and Katzenellenbogen, Nucl. Med. Biol., 24:485-498, 1997) because the charge, size, and pharmacokinetic properties of typical peptide structures do not allow diffusion across the lipid bilayer of the cell plasma membrane. This limitation has prevented peptide metal chelates from reporting the functional status or biological activity of intracellular receptors or enzymes or other homeostatic activities and intracellular targets. Although techniques and reagents for labeling antibodies and antibody fragments with metal-chelates are well known in the art (Hom and Katzenellenbogen, Nucl. Med. Biol., 24:485-498, 1997, and references therein), they target extracellular or externally oriented cell surface receptors.
Tat Proteins and Peptides
Tat is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1). The HIV-1 Tat transactivation protein is efficiently taken up by cells (Mann and Frankel, EMBO, 10:1733-1739, 1991; Vives et al., J Virol., 68:3343-3353, 1994), and low concentrations (nM) are sufficient to transactivate a reporter gene expressed from the HIV-1promoter (Mann and Frankel, EMBO, 10:1733-1739, 1991). Exogenous Tat protein is able to translocate through the plasma membrane and reach the nucleus to transactivate the viral genome (Frankel and Pabo, Cell 55:1189-1193, 1988; Ruben, et al., J Virol 63:1-8, 1989; Garcia, et al., EMBO J 7:3143, 1988; Jones, Genes Dev 11:2593-2599, 1997).
A region of the Tat protein centered on a cluster of basic amino acids is responsible for this translocation activity (Vives et al., J Biol. Chem., 272:16010-16017, 1997). Tat peptide-mediated cellular uptake and nuclear translocation have been demonstrated in several systems (Vives, et al., J Biol Chem 272:16010-16017, 1997; Jones, Genes Dev 11:2593-2599, 1997). Chemically coupling a Tat-derived peptide (residues 37-72) to several proteins results in their internalization in several cell lines or tissues (Fawell, et al., Proc Natl Acad Sci USA 91:664-668, 1994; Anderson, et al., Biochem Biophys Res Commun 194:876-8884, 1993; Fahraeus, et al., Curr Biol 6:84-91, 1996; Nagahara, et al., Nat Med 4:1449-1452, 1998). A synthetic peptide consisting of the Tat basic amino acids 48-60 with a cysteine residue at the C-terminus coupled to fluorescein maleimide translocates to the cell nucleus as determined by fluorescence microscopy (Vives et al., J Biol. Chem., 272:16010-16017, 1997). In addition, a fusion protein (Tat-NLS-xcex2-Gal) consisting of Tat amino acids 48-59 fused by their amino-terminus to xcex2-galactosidase amino acids 9-1023 translocates to the cell nucleus in an ATP-dependent, cytosolic factor-independent manner (Efthymiadis et al., J Biol. Chem., 273:1623-1628, 1998).
While the literature teaches that Tat peptide constructs and similar membrane permeant peptides readily translocate into the cytosolic and nuclear compartments of living cells, little is known regarding the cellular retention characteristics over time once the permeant peptide constructs are no longer in contact with the cell surface from the extracellular fluid spaces. Furthermore, no information is available regarding the pharmacokinetic and distribution characteristics of membrane-permeant peptides within a whole living organism, animal or human.
Apoptosis
Chemotherapeutic drugs used in the treatment of cancer are thought to interact with diverse cellular targets in conferring lethal effects on mammalian cells. Recently, anticancer agents, irrespective of their intracellular target, have been shown to exert their biological effect in target cells by triggering a common final death pathway known as apoptosis (Fulda, et al., Cancer Res 57:3823-3829, 1997; Fisher, Cell 78:539-542, 1994). Thus, there exists mounting evidence that many anticancer treatments may kill through apoptosis by activating intracellular death machinery in the target cell rather than by simply crippling various components of cellular metabolism (Fulda, et al., Cancer Res 57:3823-3829,1997; Fisher, Cell 78:539-542, 1994). In fact, the action of ionizing radiation, drug therapy, and withdrawal of physiological survival factors all appear to act as death stimuli in promoting execution of this common apoptotic pathway (Evan and Littlewood, Science 281:1317-1322, 1998; Ashkenazi and Dixit, Science 281:1305-1308, 1998). Thus, new models of resistance to therapy have begun to focus on mechanisms that antagonize execution of the apoptotic pathway.
Apoptotic stimuli can arise from the nucleus, cell membrane surface, or the mitochondria (Wyllie, Nature, 389:237-38, 1997). Ultimately, the stimuli converge on a process of activation of a family of interleukin 1xcex2-converting enzymes {(ICE)-like cysteine proteases} known as cysteine aspartases (xe2x80x9ccaspasesxe2x80x9d) (Thornberry et al., Science, 281:1312-16, 1998). Members of the caspase family are activated in apoptosis and have been shown to be necessary for programmed cell death in a number of biological systems (Yuan et al., Cell, 75:641-52, 1993; Thornberry et al., Science, 281:1312-16, 1998). The caspase gene family, defined by sequence homology, is also characterized by conservation of key catalytic and substrate-recognition amino acids (Talanian et al., J Biol. Chem., 272:9677-82, 1997). Thirteen mammalian caspases (1 through 13) have thus far been isolated, having distinct roles in apoptosis and inflammation (Thornberry et al., Science, 281:1312-16, 1998). In apoptosis, some caspases are involved in upstream regulatory events and are known as xe2x80x9cinitiators,xe2x80x9d while others are directly responsible for proteolytic cleavages that lead to cell disassembly and are known as xe2x80x9ceffectors.xe2x80x9d Evidence indicates that caspases transduce or amplify signals by mutual activation. For example, Fas-induced apoptosis is characterized by an early, transient caspase-1-like protease activity followed by a caspase-3-like activity, suggesting an ordered activation cascade (Enari et al., Nature, 380:723-26, 1996). Other data suggest that both caspase-3 and caspase-7 are activated by caspase-6 and caspase-10 (Thornberry et al., Science, 281:1312-16, 199; Fernandes-Alnemri, Proc. Natl. Acad. Sci. USA, 93:7464-69, 1996). Thus, while the activation cascade hypothesis remains to be absolutely proven (Villa et al., Trends in Biochem. Sci., 22:388-93, 1997), circumstantial evidence strongly points to caspase-3 as one key xe2x80x9ceffectorxe2x80x9d caspase, standing at the center of the execution pathway of the cell death program.
Caspases are some of the most specific of the proteases, showing an absolute requirement for cleavage after aspartic acid (Thornberry et al., Science, 281:1312-16, 1998). Recognition of at least four amino acids, amino terminal to the cleavage site, is also necessary for efficient catalysis. The preferred recognition motif differs significantly between caspases, thereby contributing to their biologically diverse functions (Talanina et al., J Biol. Chem. 272:9677-82, 1997). In addition to high specificity, caspases are also highly efficient, with Kcar/Km values  greater than 106 Mxe2x88x921 sxe2x88x921 (Thornberry et al., Science, 281:1312-16, 1998). When viewed from the perspective of a molecular target for oncological imaging, this is a key property of the caspases that allows detection of caspase activity in vivo by radiosubstrates. Another advantage of the caspases as imaging targets centers on the nature of the biochemical reaction. Because normal cells have essentially non-detectable levels of caspase activity, and once activated, the xe2x80x9ccaspase cascadexe2x80x9d amplifies reaction rates to maximal velocities (Thornberry et al., Science, 281:1312-16, 1998), the signal readout obtained by imaging is binary in character. That is, in the absence of caspase activity, the imaging signal will be low, and when activated, a highly amplified imaging signal will result. This renders the caspase-mediated enzymatic reaction essentially zero-order in situ and, therefore, independent of radiotracer concentration or specific activity, thus eliminating the complexities of first or higher order reaction rates.
Deregulation of apoptosis resulting in insufficient cell death can occur in cancer, allowing malignant tissues to grow (Thornberry et al., Science, 281:1312-16, 1998). Conversely, some diseases involve excess apoptosis, such as neurodegenerative disease, ischemia-reperfusion, graft-vs-host disease, and autoimmune disorders (Thomberry et al., Science, 281:1312-16, 1998). Accordingly, two-fold strategies for therapeutic intervention are actively underway within the pharmaceutical industry, one to selectively induce apoptosis through caspase activation, the other to inhibit caspase activity. In order to assess the treatments to alter apoptosis, an accurate means to assess apoptoic activity in vivo is needed.
Inactive pro-caspases are constitutively expressed as pro-enzymes in nearly all cells, existing in latent forms in the cell cytoplasm (Villa et al., Trends in Biochem. Sci. 22:388-93, 1997). Thus, while caspase-3 can be readily identified by Western blots, this requires biopsy material and lysis of the cells. Furthermore, activation of caspase-3 is only inferred by observation of lower molecular weight cleavage fragments on the blot. Activation of caspase-3 has also been inferred from nuclear shifts of antigen by immunohistochemical analysis of biopsy material and shown to be associated with a more favorable prognosis in, for example, pediatric neuroblastoma (Nakagawara et al., Cancer Res. 57:4578-84, 1997). However, these indirect methods only imply activation. Thus, the simple determination of the presence or absence of caspase proteins is not necessarily diagnostically useful. A method to directly and non-invasively detect and quantify the enzymatic activity of caspases in order to monitor the commitment to cell death pathway is needed. Because caspases are cytosolic enzymes, new diagnostic and therapeutic compounds are required that can readily cross cell membranes, and whose specificity is based on the presence of protease activity.
Tat Peptide Complexes
Frankel et al. (U.S. Pat. Nos. 5,804,604; 5,747,641; 5,674,980; 5,670,617; 5,652,122) discloses the use of Tat peptides to transport covalently linked biologically active cargo molecules into the cytoplasm and nuclei of cells. Frankel only discloses covalently linked cargo moieties, and does not teach or suggest the attachment of metals to Tat peptides by metal coordination complexes. Specifically, Frankel does not teach the use of peptide chelators to introduce radioimaging materials into cells. In addition, while Frankel teaches the use of cleavable coupling reagents between the Tat protein and the cargo molecule, the cleavable linkers disclosed are non-specific, such that the retention of the cargo molecule is not limited to specific cells.
Anderson et al. (U.S. Pat. Nos. 5,135,736 and 5,169,933) discloses the use of covalently linked complexes (CLCs) to introduce molecules into cells. CLCs comprise a targeting protein, preferably an antibody, a cytotoxic agent, and an enhancing moiety. Specificity is imparted to the CLC by means of the targeting protein, which binds to the surface of the target cell. After binding, the CLC is taken into the cell by endocytosis and released from the endosome into the cytoplasm. In one embodiment, Anderson discloses the use of the Tat protein as part of the enhancing moiety to promote translocation of the CLC from the endosome to the cytoplasm. In another embodiment, Anderson discloses the use of CLCs to transport radionuclides useful for imaging into cells. The complexes described by Anderson are limited in their specificity to cells that can be identified by cell surface markers. Many biologically and medically significant cellular processes, for example caspase protease activities discussed above, are not detectable with cell surface markers. In addition, the attachment of enhancing moieties to the CLC is accomplished by the use of bifunctional linkers. The use of bifunctional linkers results in the production of a heterogeneous population of CLCs with varying numbers of enhancing moieties attached at varying locations. This can lead to the production of CLCs in which the biological activity of the targeting protein, the enhancing moiety, or both, are lost. Another disadvantage of CLCs is that the number and location of linked enhancing moieties will vary with each reaction, so that a consistent product is not produced.
There is a need in the art for cell membrane-permeant peptide complexes of uniform composition, capable of delivering radionuclides, other metals, diagnostic substances such as fluorochromes, dyes, etc., and therapeutic and cytotoxic drugs into cells in a specific and selective manner. Furthermore, rapid clearance of the complexes from non-target cells and tissues of the body would facilitate and enhance the utility of such complexes in vivo.
The present inventor has surprisingly discovered that the addition of D-amino acid containing membrane-permeant peptides attached to non- or poorly permeant drugs, diagnostic substances such as oligonucleotides, peptides, peptide nucleic acids, fluorochromes, dyes, enzyme substrates, and metals useful in medical therapy, imaging, and/or diagnostics greatly increases their accumulation within cells. As shown in Example 14, this increase in accumulation is on the order of 8- to 9-fold as compared to membrane-permeant peptides comprising only naturally occurring L-amino acids. Thus, use of the D-amino acid containing membrane-permeant peptides of the present invention allows delivery of greater amounts of therapeutic or diagnostic substances to the interior of cells either in vivo or in vitro than was heretofore possible using membrane permeant peptides containing only L-amino acids.
The present inventor has also discovered that the Tat peptide and other cell membrane-permeant peptides can be used to selectively deliver non- or poorly permeant drugs, diagnostic substances such as oligonucleotides, peptides, peptide nucleic acids, fluorochromes, dyes, enzyme substrates, and metals useful in medical therapy, imaging, and/or diagnostics selectively to cells in vivo only when functional linkers are introduced into the permeant peptide construct, and has developed methods for linking these substances to Tat and other peptides for use in such methods. As illustrated in Examples 6 and 10, below, non-targeted Tat peptides, rather than being trapped inside cells and tissues indefinitely, are cleared surprisingly rapidly from body tissues when introduced into the living organism. Furthermore, non-functionalized prototypes of such complexes are rapidly excreted by the kidneys and cleared from the whole body. Thus, membrane-permeant peptides covalently linked to oligopeptides, proteins, oligonucleotides, and drugs as known previously possess rapid and ineffective biological half-times within the whole organism.
Thus, in response to this surprising and unanticipated property of D-amino acid containing permeant peptides and to improve upon the prior art, the present invention provides novel permeant peptide conjugates, complexes and methods that possess the advantage of enabling the targeted trapping of greater amounts of such compounds or fragments thereof within desired cells, tissues and organs of the intact body of living organisms. Conversely, when it is desired to increase the rates of clearance of cargo oligopeptides, proteins, oligonucleotides, metals, and drugs, the present invention also provides methods that will enhance their rates of clearance from the body.
Accordingly, in a first aspect, the present invention provides a compound comprising a cell membrane-permeant peptide; a diagnostic or pharmaceutically active substance; and a functional linker moiety linking the peptide and the diagnostic or pharmaceutically active substance, wherein the compound further comprises at least one D-amino acid and the functional linker moiety confers target cell specificity to the compound, or a pharmaceutically acceptable salt of the compound.
In a second aspect, the present invention provides a composition comprising, a compound comprising a cell membrane-permeant peptide; a diagnostic or pharmaceutically active substance; and a functional linker moiety linking the peptide and the diagnostic or pharmaceutically active substance, wherein the compound further comprises at least one D-amino acid and the functional linker moiety confers target cell specificity to the compound. The composition can further comprise a pharmaceutically acceptable carrier, excipient, or diluent.
In a third aspect, the present invention provides a kit comprising, a compound comprising a cell membrane-permeant peptide; a metal chelation ligand; and a functional linker moiety linking the peptide and the metal chelation ligand, wherein the compound further comprises at least one D-amino acid and the functional linker moiety confers target cell specificity to the compound, and a reducing agent capable of reducing a metal that can be coordinately incorporated into the metal chelation ligand.
In another aspect, the present invention provides a method for imaging cells in vivo, comprising administering to an animal a cell imaging effective amount of a compound comprising a cell membrane-permeant peptide; a chelated radionuclide or a chelated relaxivity metal; and a functional linker moiety linking the peptide and the chelated radionuclide or the chelated relaxivity metal, the functional linker confering target cell specificity to the compound, and monitoring or evaluating the location of the radionuclide or relaxivity metal within the animal. Further the compound may comprise at least one D-amino acid.
In another aspect, the present invention provides a method for imaging cells in vivo, comprising administering to an animal a cell imaging effective amount of a compound comprising a cell membrane-permeant peptide; a chelated radionuclide or a chelated relaxivity metal; and a functional linker moiety linking the peptide and the chelated radionuclide or the chelated relaxivity metal, the functional linker conferring target cell specificity to the compound, and monitoring or evaluating the location of the radionuclide or relaxivity metal within the animal. Further the compound may comprise at least one D-amino acid.
In a further aspect, the present invention provides a method for detecting cellular apoptosis in vivo, comprising administering to an animal a cellular apoptosis detecting effective amount of a compound comprising a cell membrane-permeant peptide; a diagnostic substance; and a functional linker moiety linking the peptide and the diagnostic substance, wherein the functional linker moiety comprises a caspase-reactive sequence, and monitoring the diagnostic substance within the animal. Further, the compound may contain at least one D-amino acid.
In another aspect, the present invention provides a method for detecting cellular apoptosis in vitro, comprising contacting cells or tissue in vitro with a cellular apoptosis detecting effective amount of a compound comprising a cell membrane-permeant peptide; a diagnostic substance; and a functional linker moiety linking the peptide and the diagnostic substance, wherein the functional linker moiety comprises a caspase-reactive sequence, and monitoring the diagnostic substance within the cells or tissue. Further, the compound may contain at least one D-amino acid.
In yet another aspect, the present invention provides a method for detecting an enzyme in a cell, comprising contacting the cell with an enzyme detecting effective amount of a compound comprising a cell membrane-permeant peptide; a diagnostic substance; a functional linker moiety linking the peptide and the diagnostic substance, wherein the functional linker moiety comprises a sequence reactive with the enzyme; removing unreacted compound from the locus of the cell so that the signal to noise ratio is sufficient for diagnostic purposes; and monitoring the presence of the diagnostic substance in the cell. Such monitoring can be performed quantitatively, and the cell can be present within a living animal. Furthermore, the enzyme can be one that is characteristically associated with a disease, condition, or disorder. Further, the compound may contain at least one D-amino acid.
In yet another aspect, the present invention provides a method for diagnosing the presence of a disease, condition, or disorder in an animal, comprising administering to the animal a diagnostically effective amount of a compound comprising a cell membrane-permeant peptide; a diagnostic substance; a functional linker moiety linking the peptide and the diagnostic substance, wherein the functional linker moiety confers target cell specificity to the compound, and which comprises a sequence reactive with an enzyme indicative or characteristic of the disease, condition, or disorder, and monitoring the diagnostic substance within the animal. By way of example, the disease, condition, or disorder can be a cancer such as a central nervous system tumor, breast cancer, liver cancer, lung cancer, head cancer, neck cancer, a lymphoma, or a melanoma. Further, the compound may contain at least one D-amino acid.
In still another aspect, the present invention provides a method of assessing the effectiveness of cancer therapy, comprising administering to an animal undergoing cancer therapy a diagnostically effective amount of a compound comprising a cell membrane-permeant peptide; a diagnostic substance; and a functional linker moiety linking the peptide and the diagnostic substance, wherein the functional linker moiety confers target cell specificity to the compound, and which comprises a caspase-reactive sequence, and monitoring the diagnostic substance within the animal. Such monitoring can be performed quantitatively. Furthermore, the method can be repeated at intervals during the cancer therapy, and the quantity of the diagnostic substance detected within the animal at each interval can be compared to the quantity of the diagnostic substance detected at previous intervals to determine the effectiveness of the therapy. In addition, the compound may contain at least one D-amino acid.
In yet another aspect, the present invention provides a method of delivering a pharmaceutically active substance to a cell, comprising contacting the cell with an effective amount of a compound comprising a cell membrane-permeant peptide; a pharmaceutically active substance; and a functional linker moiety linking the peptide and the pharmaceutically active substance, wherein the compound further comprises at least one D-amino acid and the functional linker moiety confers target cell specificity to the compound.
In another aspect, the present invention provides a method of treating, inhibiting, or preventing a disease, condition, or disorder responsive to treatment with a pharmaceutically active substance in an animal, comprising administering to the animal a pharmaceutically effective amount of a compound comprising a cell membrane-permeant peptide; a pharmaceutically active substance; and a functional linker moiety linking the peptide and the pharmaceutically active substance, wherein the compound further comprises at least one D-amino acid and the functional linker moiety confers target cell specificity to the compound.
In another aspect, the present invention provides a method for selectively destroying cells expressing a selected enzyme activity, comprising contacting the cells with a cell-destroying effective amount of a compound comprising a cell membrane-permeant peptide; a cytotoxic substance; and a functional linker moiety linking the peptide and the cytotoxic substance, wherein the compound further comprises at least one D-amino acid and the functional linker moiety confers target cell specificity to the compound.
In yet another aspect, the present invention provides a method for assessing the effect of a drug in altering the expression or activity of an enzyme in a target cell, comprising contacting the target cell with a diagnostically effective amount of a compound comprising a cell membrane-permeant peptide; a diagnostic substance; a functional linker moiety linking the peptide and the diagnostic substance, wherein the functional linker moiety confers target cell specificity to the compound, and which comprises a sequence capable of interacting with the enzyme so as to release the diagnostic substance from the compound into the interior of the cell; clearing unreacted compound from the locus of the cell so that the signal to noise ratio is sufficient for diagnostic purposes; and monitoring or evaluating the diagnostic substance in the target cell. Such monitoring can be performed quantitatively, and the target cell can be present within a living animal. Furthermore, the enzyme can be associated with a disease, condition, or disorder. In addition, the compound may further comprise at least one D-amino acid.
In yet another aspect, the present invention provides a method for detecting the expression of a nucleic acid sequence, which can be DNA or RNA, encoding an enzyme, a receptor, or a binding protein introduced into a cell, comprising contacting the cell with a compound comprising a cell membrane-permeant peptide; a diagnostic substance; a functional linker moiety linking the peptide and the diagnostic substance, wherein the functional linker moiety confers target cell specificity to the compound, and which comprises a sequence capable of interacting with the enzyme, receptor, or binding protein so as to selectively retain the diagnostic substance in the cell, and monitoring the diagnostic substance in the cell. Further, the compound may comprise at least one D-amino acid.
Further scope of the applicability of the present invention will become apparent from the detailed description and drawings provided below. However, it should be understood that the following detailed description and examples, while indicating preferred embodiments of the invention, are given by way of illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.